The present invention involves the design, synthesis and use of synthetic analogs of the luteinizing hormone releasing hormone (LHRH). An important achievement involved synthesis of analogs which functioned as antagonists of LHRH, were adequately potent to inhibit ovulation and allowed the release of only negligible amounts of histamine. Since there was no way of reliably forecasting the structure of an antagonist having high potency and very low histamine release, it was necessary to explore diverse approaches to discover a combination of structural features which would yield an antagonist of LHRH having high potency for ovulation inhibition and very low activity for histamine release.
Various peptides such as substance P, vasoactive intestinal peptide, gastrin, somatostatin, as well as others, are well known to cause the release of histamine from mast cells. These cells are in many tissues, such as skin, lung and mesentery, gingiva, etc. Most cells have granules containing histamine and other mediators of inflammation which can be released by peptides to cause capillary dilation and increased vascular permeability. When it was noted that an antagonist of LHRH, for example [Ac--D--2--Nal.sup.2, D--4--F--Phe.sup.2, D--TrP.sup.3, D--Arg.sup.6 ]--LHRH, caused edema of the face and extremities when it was administered to rats, it appeared likely that such antagonists, if administered to human subjects as a contraceptive agent, would cause serious edema of the face and elsewhere in the human body. Such side effects would likely prevent the administration of such antagonists to human subjects.
The histamine-containing leukocyte is a basophile which can also release histamine when stimulated by many of the same peptides mentioned above. Basophiles differ biochemically from mast cells and such differences may allow for both predictable and unpredictable histamine release in response to antagonists of LHRH. An antagonist of LHRH, to be used clinically to prevent ovulation, should not significantly release amounts of histamine from either mast cells or basophiles.
The discovery of the side effects such as the edematogenic and anaphylactoid actions of LHRH antagonists made desireable the discovery of new LHRH antagonists which prevented ovulation but did not release significant histamine. These undesireable side effects have been observed in rats, and it is likely that the Food and Drug Administration would not allow the testing of such antagonists in human subjects.
Karten et al. (4), have reviewed available knowledge on the structural characteristics for potent histamine release by antagonists of LHRH. Some of the most important findings are as follows. A most potent LHRH antagonist in triggering histamine release in vitro involved a combination of strongly basic D-amino acid side chains (Arg or Lys) at position 6 and in close proximity to Arg.sup.8, and a cluster of hydrophobic aromatic amino acids at the N-terminus. Thus, there is no specific amino acid of the ten amino acids which is solely responsible for histamine release. On the contrary, structural features ranging from the N-terminus (the amino acids in the first few positions, 1-4, etc.), and basic amino acids toward the C-terminus (positions 6 and 8) somehow participate in histamine release. Even D--Ala in position 10 has some influence on histamine release, the rationale for which is unclear. By themselves, two basic side chains in close proximity, as in positions 6 and 8, are insufficient alone to impart high release of histamine. The cluster of hydrophobic amino acids at the N-terminus is insufficient alone for high histamine releasing activity. Even a hexapeptide fragment has revealed moderate histamine releasing potency. There seems to be no correlation between antiovulatory potency and histamine release of these antagonists, in vitro.
In perspective, much of the entire chain of such decapeptide antagonists may have influence on histamine release. The same perspective appears to be true, but to different degrees, for high antiovulatory activity. These LHRH antagonists are usually decapeptides which indicates that there are ten variables to adjust for a desired anti-ovulatory activity and ten variables to adjust for eliminating histamine releasing activity. There are even further variations for each of these twenty variables, the number of possible peptides to design, synthesize and assay becoming incalculable. Presumably, some of the ten variables may be independent for anti-ovulatory activity and histamine releasing activity while some variables may overlap for these two biological activities. This situations presents a extraordinary difficulties to solve before an antagonist of high potency for anti-ovulation and very low potency for histamine release could be produced.
Diverse structural changes and combinations of the ten amino acids followed by assays of both anti-ovulation and histamine release activities should be performed in the hope that a potent antagonist essentially free of side effects would be discovered. The synthesis of new amino acids to introduce into the decapeptide chains should also be explored since the commonly available amino acids might not suffice.